Embed Size (px)
Citation preview
Charles Darwin University
Circumsporozoite-specific T cell responses in children vaccinated with RTS,S/AS01 Eand protection against P falciparum clinical malaria
Olotu, Ally; Moris, Philippe; Mwacharo, Jedidah; Vekemans, Johan; Kimani, Domtila;Janssens, Michel; Kai, Oscar; Jongert, Erik; Lievens, Marc; Leach, Amanda; Villafana, Tonya;Savarese, Barbara; Marsh, Kevin; Cohen, Joe; Bejon, PhilipPublished in:PLoS One
DOI:10.1371/journal.pone.0025786
Published: 01/01/2011
Document VersionPublisher's PDF, also known as Version of record
Link to publication
Citation for published version (APA):Olotu, A., Moris, P., Mwacharo, J., Vekemans, J., Kimani, D., Janssens, M., Kai, O., Jongert, E., Lievens, M.,Leach, A., Villafana, T., Savarese, B., Marsh, K., Cohen, J., & Bejon, P. (2011). Circumsporozoite-specific T cellresponses in children vaccinated with RTS,S/AS01 E and protection against P falciparum clinical malaria. PLoSOne, 6(10), 1-11. [e25786]. https://doi.org/10.1371/journal.pone.0025786
General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.
• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal
Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.
Download date: 29. Apr. 2020
Circumsporozoite-Specific T Cell Responses in ChildrenVaccinated with RTS,S/AS01E and Protection againstP falciparum Clinical MalariaAlly Olotu1, Philippe Moris2, Jedidah Mwacharo1, Johan Vekemans2, Domtila Kimani1, Michel Janssens2,
Oscar Kai1, Erik Jongert2, Marc Lievens2, Amanda Leach2, Tonya Villafana3,4, Barbara Savarese3, Kevin
Marsh1,5, Joe Cohen2, Philip Bejon1,5*
1 Kenya Medical Research Institute/ Wellcome Trust Programme, Centre for Geographic Medicine Research, Coast, Kilifi, Kenya, 2 GlaxoSmithKline Biologicals, Rixensart,
Belgium, 3 PATH Malaria Vaccine Initiative (MVI), Bethesda, Maryland, United States of America, 4 MedImmune, LLC, Gaithersburg, Maryland, United States of America,
5 Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Abstract
Background: RTS,S/AS01E is the lead candidate pre-erythrocytic malaria vaccine. In Phase IIb field trials the safety profile wasacceptable and the efficacy was 53% (95%CI 31%–72%) for protecting children against clinical malaria caused by P.falciparum. We studied CS-specific T cell responses in order to identify correlates of protection.
Methods and Findings: We used intracellular cytokine staining (for IL2, IFNc, and TNFa), ex-vivo ELISPOTs (IFNc and IL2) andIFNc cultured ELISPOT assays to characterize the CS-specific cellular responses in 407 children (5–17 months of age) in aphase IIb randomized controlled trial of RTS,S/AS01E (NCT00380393). RTS,S/ AS01E vaccinees had higher frequencies of CS-specific CD4+ T cells producing IFNc, TNFa or IL2 compared to control vaccinees. In a multivariable analysis TNFa+ CD4+ Tcells were independently associated with a reduced risk for clinical malaria among RTS,S/AS01E vaccinees (HR = 0.64, 95%CI0.49–0.86, p = 0.002). There was a non-significant tendency towards reduced risk among control vaccinees (HR = 0.80, 95%CI0.62–1.03, p = 0.084), albeit with lower CS-specific T cell frequencies and higher rates of clinical malaria. When data fromboth RTS,S/AS01E vaccinees and control vaccinees were combined (with adjusting for vaccination group), the HR was 0.74(95%CI 0.62–0.89, p = 0.001). After a Bonferroni correction for multiple comparisons (n-18), the finding was still significant atp = 0.018. There was no significant correlation between cultured or ex vivo ELISPOT data and protection from clinical malaria.The combination of TNFa+ CD4+ T cells and anti-CS antibody statistically accounted for the protective effect of vaccinationin a Cox regression model.
Conclusions: RTS,S/AS01E induces CS-specific Th1 T cell responses in young children living in a malaria endemic area. Thecombination of anti-CS antibody concentrations titers and CS-specific TNFa+ CD4+ T cells could account for the level ofprotection conferred by RTS,S/AS01E. The correlation between CS-specific TNFa+ CD4+ T cells and protection needsconfirmation in other datasets.
Citation: Olotu A, Moris P, Mwacharo J, Vekemans J, Kimani D, et al. (2011) Circumsporozoite-Specific T Cell Responses in Children Vaccinated with RTS,S/AS01E
and Protection against P falciparum Clinical Malaria. PLoS ONE 6(10): e25786. doi:10.1371/journal.pone.0025786
Editor: Lorenz von Seidlein, Menzies School of Health Research, Australia
Received July 11, 2011; Accepted September 9, 2011; Published October 6, 2011
Copyright: � 2011 Olotu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project has been funded by PATH Malaria Vaccine Initiative (MVI). Prof. Marsh is supported by the Wellcome Trust. Philip Bejon is supported by theBiomedical Research Centre in Oxford. Two authors (TV and BS) are MVI employees and were involved in trial design and writing the report. Dr. Villafana was anemployee of MVI throughout the duration of the study, but has since moved to MedImmune, LLC. MedImmune, LLC had no role in the study design, datacollection, analysis, decision to publish, preparation of the manuscript or any other aspect of the study. The Wellcome Trust and Biomedical Research Centre hadno role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. MVI had no other role in data collection or analysis, andthe decision to publish was covered by a pre-study agreement.
Competing Interests: Dr. Leach, Mr. Lievens, Dr. Vekemans, Dr. Jongert, and Dr. Cohen report being employees of GlaxoSmithKline Biologicals; Dr. Leach,Dr. Vekemans and Dr. Cohen report owning shares in GlaxoSmithKline. Dr. Cohen reports being listed as an inventor of patents covering the RTS,S malaria vaccinecandidate, which include patents and patent applications related to International Patent Application Publication Nos. WO93/10152, WO2006/029887, WO2007/003384, WO2009/021931 and WO2009/080715, the rights to which have been assigned to GlaxoSmithKline Biologicals. Ms. Savarese, and Dr. Villafana reportbeing employees of MVI, which supports the development and testing of several malaria vaccines (i.e. Ad26/35-CSP (Crucell), PvRII (ICGEB), whole irradiatedsporozoites (Sanaria), MSP2 (LaTrobe/QIMR), AMA1 (NIH/QIMR) and RTS,S/AS01). Dr. Villafana was an employee of MVI throughout the duration of the study, buthas since moved to MedImmune, LLC. No other potential conflict of interest relevant to this article was reported. This does not alter the authors’ adherence to allthe PLoS ONE policies on sharing data and materials.
* E-mail: [email protected]
Introduction
RTS,S is the lead candidate pre-erythrocytic malaria vaccine
[1]. The vaccine antigen consists of 19 copies of the central
tandem repeats and C-terminal region of the P. falciparum
circumsporozoite protein (CS) fused to hepatitis B surface antigen
(HBsAg), and co-expressed with unfused HBsAg in Saccharomyces
cerevisiae cells. The two proteins spontaneously assemble in the
yeast cells to form virus-like particles. The RTS,S antigen has been
tested with two different alternative Adjuvant Systems: AS02 or
PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25786
AS01. Both Adjuvant Systems contain the immunostimulants
monophosphoryl lipid A (MPLH) and QS21, formulated either
with an oil-in-water emulsion (AS02) or with liposomes (AS01).
Formulated in either Adjuvant System, the RTS,S antigen
induces high concentrations of anti-circumsporozoite protein (CS)
antibodies [2,3,4,5,6,7]. Correlations between anti-CS concentra-
tions and protection against infection were statistically significant
on experimental challenge with P. falciparum in malaria naı̈ve
adults [7], of borderline significance on natural challenge of semi-
immune adults [4], and significant on natural challenge of children
in a malaria endemic area [8]. Anti-CS titers did not correlate with
protection against clinical malaria episodes in children [4,9], but
we recently identified a non-linear relationship between concur-
rent (rather than peak) anti-CS titers and protection from clinical
malaria in children [10].
CD4+ T cell responses to pre-erythrocytic antigens prevent
intra-hepatocytic parasites developing in both human and mouse
studies [11,12]. Potential mechanisms include TNFa induced
apoptosis [13] or inhibition of parasite growth [14] and IFNcinduced NO production [15]. RTS,S-induced cell mediated
immune responses have been assessed using proliferation assays,
cytokine production on cell culture, intracellular cytokine staining
and flow-cytometry, and ex-vivo and cultured ELISPOT assays
[16,17].
RTS,S/AS immunization induces a CD4+ T cell response but
little or no detectable CD8+ T cell response [7,18,19,20,21]. Sun
et al observed IFNc-producing CD8+ T cells, but only after cells
were stimulated for 10–14 days in vitro [22]. Barbosa et al reported
CD8+ T cell responses after 42 hours in vitro stimulation on
comparing RTS,S/AS02 vaccinees with control vaccinees at 10
weeks, but not at 4 weeks, post immunization [23].
The frequency of poly-functional CD4+ T cells identified by
intracellular cytokine staining (ICS) correlated with protection
from P. falciparum infection after experimental challenge in adults
[7,24]. In a field study, Reece et al reported a correlation between
protection against re-infection and cultured IFNc ELISPOT
assays using a single conserved T cell epitope from the CS protein
[20]. However, this analysis was not adjusted for anti-CS titers,
and did not include ICS studies. A borderline correlation between
single cytokine ICS results and protection from P. falciparum
infection was shown in a field study in infants [23].
In order to examine associations with protection against clinical
malaria, we assessed the CS-specific cellular immune responses in
447 children using ICS, ex vivo IFNc and IL2 ELISPOT, and
cultured IFNc ELISPOT assays in a phase II b randomized
clinical trial of RTS,S/AS01E versus control, in which we
observed 53% (95%CI 31%–72%) protection against clinical
malaria [25]. The blood volumes sampled in children prevented us
from using an ICS assay previously reported in adult studies [7],
but a whole blood ICS assay requiring smaller blood volumes has
been developed and used in two phase II trials in Ghana [26] and
Gabon [27]. These studies showed that the vaccine induced CD4+IL2, TNFa or IFNc producing cells, but CD40L was not
detectable using the whole blood assay for children in Sub-
Saharan Africa. We therefore did not include CD40L staining in
the assay for our study.
The qualification of correlates of immunity and surrogates of
protection has been recently reviewed [28,29]. The Prentice
criteria require that: a) vaccination predicts protection; b)
vaccination predicts the potential surrogate; c) the surrogate
predicts protection among vaccinees and d) that the surrogate
accounts for all the effect of vaccination [30]. If vaccination is an
independent predictor of outcome after including the potential
surrogate in the analysis, this suggests that other mechanisms are
involved. On the other hand, if including the potential surrogate in
analysis removes vaccination as a predictor, this is consistent with
the effect of vaccination being mediated by the surrogate marker.
Methods
The study protocol and its amendments received ethical and
scientific approval from Kenya Medical Research Institute
National Ethics Committee, National Institute for Medical
Research of Tanzania, the Oxford Tropical Research Ethics
Committee, the London School of Hygiene and Tropical
Medicine Ethics committee and the Western Institutional Review
Board in Seattle. The study was conducted in accordance with the
Helsinki Declaration of 1964 (revised 1996) and Good Clinical
Practice guidelines and was overseen by an Independent data
monitoring committee and local safety monitors. Written informed
consent was obtained using approved Swahili or Giriama consent
forms. Illiterate parents thumb printed the consent form which
was countersigned by an independent, literate witness.
We conducted a randomized controlled trial to evaluate the
efficacy and safety of RTS,S/AS01E against clinical malaria
episodes due to P. falciparum infection in Kilifi, Kenya and
Korogwe, Tanzania. There were 894 children between the two
sites, of which the 447 children enrolled in Kilifi were assessed for
vaccine induced cellular immunity using ICS and ELISPOT.
Details on randomization, immunization and surveillance have
been published previously [25]. In Kilifi, 447 children 5–17 months
old were randomized and received either RTS,S/AS01E or rabies
vaccine in a 1:1 ratio according to 0, 1, 2 month schedule. Both
vaccines were given intramuscularly in the left deltoid. The primary
end point was clinical malaria, defined as the presence of fever
(axillary temperature $37.5uC) and P. falciparum parasitaemia
$2500/mL. Active and passive surveillance for malaria was
conducted by field workers and study personnel at local dispensaries.
Children were vaccinated between March and August 2007.
Blood was taken for immunological studies before vaccination, one
month post dose 3, then on March 2008 irrespective of the time of
recruitment (i.e. between 4 and 10 months post dose 3, mean 8
months), 12 months post dose 3 and in October 2008 irrespective
of time of recruitment, (i.e. between 12 and 18 months post dose 3,
mean 15 months). Peak malaria transmission was between May
and August 2008.
CS antibody measurementAntibodies to the P. falciparum circumsporozoite protein (CS) tandem
repeat epitope were assessed by ELISA at the Center for Vaccinology,
Ghent University Hosptial, Belgium. Results were reported in EU/
mL. Plates were adsorbed with the recombinant antigen R32LR that
contained the sequence [NVDP(NANP)15]2LR [31].
PeptidesA set of 32 15-mer, peptides were used, overlapping by 11
amino acids to cover the full length of the CS antigen used in the
vaccine (3D7 strain). All these peptides were used in a single pool
for the ICS studies, but they were divided into three pools for
ELISPOT studies, namely; a) the conserved region including the
NANP repeats, b) the variant TH2R region and c) the variant
TH3R region and conserved CS.T3T region (Table 1).
ELISPOT assaysPeripheral blood mononuclear cells (PBMC) were separated
and incubated in RPMI medium (Sigma-Aldrich) with 10%
Human AB serum. We used Millipore MAIP S45 plates and
MabTech antibodies for ELISPOT assays according to the
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25786
manufacturer’s instructions. For ex vivo ELISPOT assays (IFNcand IL2), 26105 per well of freshly isolated PBMCs were
incubated in 100ml final volume at 2.5 mg/ml circumsporozoite
antigen peptides (see table 1) for 18–20 hours before developing
the plates. The positive and negative controls were 20 mg/ml
Phytohaemaglutinin (Sigma-Aldrich) and media alone, respective-
ly. For cultured ELISPOTs, 1x106 PBMC were incubated in
0.5 mls of 10mg/ml/peptide of pooled peptides in a 24-well plate.
On days 3 and 7, 250ml of culture supernatant was replaced with
250ml culture medium containing 20 IU/ml recombinant IL 2.
On day 9, the cells were washed three times and left overnight
before an ELISPOT assay (IFNc only) was done according to the
method used for ex vivo ELISPOTs. Spot forming cell numbers
were counted by ELISPOT plate reader (Autoimmun Diagnos-
tika, version 3.0).
ELISPOT analysisELISPOT wells were assayed in duplicate, and the final result
was the mean of two wells. The negative control well result was
subtracted from each peptide well. ELISPOTs failed quality
control if the negative control well had more than 25 spots or the
positive control had less than 50 spots. The results from the three
peptide pools were added to calculate total responses. Results are
presented as number of spots per million incubated PBMC.
Whole blood ICS assayWhole blood was stimulated in Kilifi within 2 hours of being
drawn. 350 ml of whole blood plus 100 ml of phosphate buffered
saline (PBS) was incubated in three different 15 ml Falcon tube,
with 1 mg/ml of anti-CD28 anti-CD49d monoclonal antibodies
(supplied by BD). After 2 hours, Brefeldin A was added to a final
concentration of 1 mg/ml and incubation was continued overnight
at 37 ˚C 6 1- CO2 5 to 7%. EDTA was then added to a final
concentration at 5 mM, and after 15 minutes 1 ml FACS lysing
solution (BD). The positive control was stimulated using
staphylococcal enterotoxin B (SEB) and negative control was
PBS without peptides. Circumsporozoite antigen peptides were
added to the third tube to a final concentration of 1 mg/ml (see
Table 1). The cells were then washed in PBS and re-suspended in
PBS with 10% DMSO and stored at –70 ˚C for transport to GSK
in Rixensart. In GSK, cells were thawed, washed and stained with
alexa-fluor 700 conjugated anti-CD3 (Pharmingen), peridinin-
chlorophyll (PerCP)-conjugated anti-CD4 (BD Biosciences) and
allophycocyanin (APC)-H7 conjugated anti-CD8 antibodies (BD
Biosciences). Cells were fixed and permeabilized using the
Cytofix/Cytoperm buffer kit (Pharmingen), and stained with
APC conjugated anti-IL-2 (Pharmingen), fluorescein-isothiocya-
nate (FITC)-conjugated anti-IFN-c (Pharmingen) and phycoery-
thrin (PE) cyanin–7 (Cy7)-conjugated anti-TNFa (Pharmingen).
Cells were washed, re-suspended in fetal-calf-serum (FCS)-
containing phosphate buffered saline (PBS) and analyzed on a
BDTM LSR II flow cytometer (BD Biosciences). Events were
counted using the automatic gating on the FACSDiva software
(BD Biosciences). Conventional rules were used to gate on single
cells, then the lymphocyte subset based on forward and side
scatter. CD3 and CD4/CD8 positive cells and then cytokine
expression was classified into positive/negative using FACSDiva
software. An example of the output with gating shown is given in
Supporting Information S1. We required at least 10,000 CD4 +events and 5,000 CD8+ events. Acquisition was stopped when
75,000 CD4+ events had been acquired, and we acquired more
than 50,000 CD4+ events for the majority of samples (.90%).
Results from antigen-stimulated cultures were not excluded from
analysis on the basis of positive/negative control results, in the
absence of established criteria. Data are represented as background
subtracted CS-specific events per million CD4+ or CD8+ T cells.
Assays were conducted according to sample availability, since
blood samples were limited to 5 mls. In order of priority, the
assays conducted were; ICS, IFNc ex vivo ELISPOT, IL2 ex vivo
ELISPOT and cultured ELISPOT. Samples were processed
within 3 hours of being taken. ICS samples were stored for 3 to
4 months at –70 ˚C before staining. The samples were processed
during the double-blind phase of the study.
Statistical analysisGeometric mean responses are calculated and a Student’s T test
was performed on log-transformed values to compare between
vaccination groups. A paired T test on log-transformed values was
used to compare time-courses, and correlations between assays
were examined using Pearson’s product moment calculation on
Table 1. Peptide pools.
Peptide Sequence
NANP and conserved region peptides pool
Pept 1 MMAP DPNANPNANPN
Pept 2 NANP NANPNANPNAN
Pept 3 DPNA NPNANPNKNNQ
Pept 4 NPNA NPNKNNQGNGQ
Pept 5 NPNK NNQGNGQGHNM
Pept 6 NNQG NGQGHNMPNDP
Pept 7 NGQG HNMPNDPNRNV
Pept 8 HNMP NDPNRNVDENA
Pept 9 NDPN RNVDENANANS
Pept 10 RNVD ENANANSAVKN
Pept 11 ENAN ANSAVKNNNNE
TH2R region peptides pool
Pept 12 ANSA VKNNNNEEPSD
Pept 13 VKNN NNEEPSDKHIK
Pept 14 NNEE PSDKHIKEYLN
Pept 15 PSDK HIKEYLNKIQN
Pept 16 HIKE YLNKIQNSLST
Pept 17 YLNK IQNSLSTEWSP
Pept 18 IQNS LSTEWSPCSVT
Pept 19 LSTE WSPCSVTCGNG
TH3R/CS.T3T region peptides pool
Pept 20 WSPC SVTCGNGIQVR
Pept 21 SVTC GNGIQVRIKPG
Pept 22 GNGI QVRIKPGSANK
Pept 23 QVRI KPGSANKPKDE
Pept 24 KPGS ANKPKDELDYA
Pept 25 ANKP KDELDYANDIE
Pept 26 KDEL DYANDIEKKIC
Pept 27 DYAN DIEKKICKMEK
Pept 28 DIEK KICKMEKCSSV
Pept 29 KICK MEKCSSVFNVV
Pept 30 MEKC SSVFNVVNSSI
Pept 31 KCSS VFNVVNSSIGL
All three peptide pools were combined for the ICS assay. The pools were usedseparately for the ex vivo and plating out of the cultured ELISPOT assay.doi:10.1371/journal.pone.0025786.t001
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25786
log-transformed values. Cox regression for the primary endpoint
(clinical malaria with P. falciparum density $2500/mL) was adjusted
for age at first vaccination, village, distance from the health facility,
bed net use and anti-circumsporozoite (CS) antibody levels by
dichotomizing concurrent anti-CS titers at 40 EU/mL [10].
Cellular responses were analyzed as time-varying covariates,
applying the result from the time of the most recent clinic visit.
A Bonferroni correction was subsequently calculated for the
independently significant explanatory variables. Responses were
log transformed to produce normal distributions before inclusion
in the Cox regression models. Analysis was conducted on the
According To Protocol vaccinees. STATA version 10 was used.
Results
Blood samples were processed from 407 children. Data were
acquired from 1,066 ICS assays (from three different clinic visits),
660 cultured ELISPOTs (from four different clinic visits), 780 ex
vivo ELISPOTs for IFNc (from three clinic visits) and 453 ex vivo
ELISPOTs for IL2 production (from 3 clinic visits). 56 (8%), 12
(2%) and 21 (5%) assays failed quality control criteria for positive
and negative controls for cultured, and ex vivo IFNc and ex vivo IL2
ELISPOTs, respectively. For ICS assay, the results from the
positive control were at least 100 cells per million above the
negative control for 1045 (98.0%), 1057 (99.1%) and 1055 (99.0%)
for IFNc, IL2 and TNFa, respectively.
The geometric mean responses to the negative control were 75,
165 and 159 cells per million for IFNc, IL2 and TNFa ICS results
respectively, and average responses to positive control were 3,768,
18,895 and 3,454 cells per million. The mean responses to CS
antigen vary by timepoint and by vaccination group, but the
ranges were 11 to 25, 10 to 681 and 8 to 426 for IFNc, IL2 and
TNFa, respectively. There was no variation in responses to control
by time point (p = 0.15, p = 0.15, p = 0.6) or by vaccination group
at the first timepoint post vaccination (p = 0.4, p = 0.36 and
p = 0.39). An example of the flow cytometry analysis is shown in
Figure 1.
Vaccine induced anti-CS T cell responses: ICS assaysCD4+ and CD8+ anti-CS T cell responses were detected in
both vaccination groups using ICS. There were no significant
differences between the groups pre-vaccination. Vaccination with
RTS,S/AS01E induced CD4+ but no CD8+ anti-CS T cell
responses. The strongest responses were seen for IL2 producing
CD4 T cells at one month post vaccination (a mean of 681 cells
per million, 95%CI 585–792), followed by TNFa (426 cells per
million, 95%CI 362–502), and weak IFNc responses (25 cells per
million, 95%CI 18–34) (Table 2, Figure 2). These levels
Figure 1. An example plot of FACS data acquired following intra-cellular cytokine staining is shown for negative control (mediumonly), positive control (i.e. staphylococcal enterotoxin B, SEB) and CS peptides.doi:10.1371/journal.pone.0025786.g001
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25786
corresponded to 3.2 fold, 2.3 fold and 1.9 fold increases for IL2,
TNFa and IFNc, respectively.
ELISPOT assaysThree different peptide pools were used for the ELISPOT
assays, allowing a more detailed analysis of immunogenicity.
Cultured ELISPOT results were higher among RTS,S/AS01E
vaccinees than among rabies vaccinees at 1 month and 6.5 months
post vaccination, but not at 12 months. IFNc ex vivo ELISPOT
results did not vary by vaccination group at any timepoint. IL2 ex
vivo ELISPOT responses were significantly higher in RTS,S/
AS01E vaccinees at 1 month post vaccination, but not at 6.5
months (Table 2) compared with rabies vaccinees.
For both the cultured IFNc ELISPOT and ex vivo IL2
ELISPOT, the vaccine induced cellular responses were limited
to two peptide pools (i.e. TH2R and TH3R/CS.T3T pools, Table
1). No responses were detected to the third peptide pool (NANP
and conserved region peptides; Figure 3).
Table 2. Geometric means of CMI assays by clinic visit and byvaccination group.
Visit Rabies RTS,S/AS01E
Mean(95%CI) N
Mean(95%CI) N p
ICS: CD4+ve cells: IFNg
Screen 11(8–14) 197 12(9–16) 182 0.6
Vac+1 mth 13(10–18) 182 25(18–34) 170 0.01*
Vac+12 mths 11(7–15) 167 20(14–29) 168 0.009**
ICS: CD4+ve cells: IL2
Screen 103(84–127) 197 94(76–117) 182 0.52
Vac+1 mth 212(183–245) 182 681(585–792) 170 2610–13**
Vac+12 mths 10(7–14) 167 102(73–142) 168 9610–19**
ICS: CD4+ve cells: TNFa
Screen 86(69–108) 197 81(64–102) 182 0.69
Vac+1 mth 182(156–214) 182 426(362–502) 170 1610–08**
Vac+12 mths 8(6–11) 167 48(34–68) 168 6610–12**
ICS: CD8+ve cells: IFNg
Screen 12(9–17) 178 7(5–10) 175 0.028#
Vac+1 mth 19(12–28) 168 19(12–28) 156 0.99
Vac+12 mths 10(6–15) 137 11(7–17) 127 0.73
ICS: CD8+ve cells: IL2
Screen 156(128–191) 178 190(155–233) 175 0.24
Vac+1 mth 200(164–246) 168 215(175–266) 156 0.64
Vac+12 mths 9(6–13) 137 20(13–32) 127 0.004**
ICS: CD8+ve cells: TNFa
Screen 49(36–68) 178 59(43–81) 175 0.43
Vac+1 mth 133(106–167) 168 162(127–205) 156 0.26
Vac+12 mths 15(10–24) 137 16(10–25) 127 0.91
IFNg cultured ELISPOT: NANP and conserved region
Screen 27(21–35) 72 33(26–43) 70 0.22
Vac+1 mth 32(27–38) 86 28(24–32) 109 0.26
Vac+6.5 mths 29(25–35) 82 26(22–30) 122 0.34
Vac+12 mths 31(23–42) 55 27(21–36) 64 0.54
IFNg cultured ELISPOT: TH2R region
Screen 33 (26–43) 72 36 (28–48) 70 0.66
Vac+1 mth 34(25–47) 86 66(50–88) 109 3610–4**
Vac+6.5 mths 30(22–41) 83 60(47–78) 122 2610–4**
Vac+12 mths 26(20–34) 55 39(30–51) 64 0.023*
IFNg cultured ELISPOT: TH3R/ CS.T3T region
Screen 31 (23–41) 72 37 (28–48) 70 0.38
Vac+1 mth 30(23–40) 86 55(43–70) 109 3610–4**
Vac+6.5 mths 32(23–44) 83 58(45–76) 122 0.003**
Vac+12 mths 33(24–46) 55 36(26–48) 64 0.79
IFNg cultured ELISPOT: All CS peptides summed
Screen 75 (58–97) 72 90 (69–117) 70 0.7
Vac+1 mth 88(66–117) 86 151(117–195) 109 0.002**
Vac+6.5 mths 81(59–110) 82 145(113–187) 122 0.002**
Vac+12 mths 83(59–115) 55 104(76–141) 64 0.33
IFNg ex vivo ELISPOT: NANP and conserved region
Screen 15(13–17) 152 14(12–16) 137 0.59
Vac+1 mth 15(13–18) 100 16(13–19) 104 0.83
Visit Rabies RTS,S/AS01E
Mean(95%CI) N
Mean(95%CI) N p
Vac+6.5 mths 13(12–14) 145 12(11–13) 142 0.21
IFNg ex vivo ELISPOT: TH2R region
Screen 15(13–17) 152 14(12–16) 137 0.56
Vac+1 mth 16(13–19) 100 18(15–22) 104 0.23
Vac+6.5 mths 14(13–16) 145 14(13–17) 142 0.96
IFNg ex vivo ELISPOT: TH3R/ CS.T3T region
Screen 18(15–20) 152 17(15–20) 137 0.79
Vac+1 mth 19(15–23) 100 21(17–25) 104 0.43
Vac+6.5 mths 13(11–15) 144 15(13–17) 141 0.23
IFNg ex vivo ELISPOT: All CS peptides summed
Screen 40(35–46) 152 39(34–45) 137 0.69
Vac+1 mth 44(36–54) 100 48(40–59) 104 0.5
Vac+6.5 mths 35(31–40) 144 35(31–40) 141 0.94
IL2 ex vivo ELISPOT: NANP and conserved region
Screen 15(13–18) 107 15(12–18) 89 0.8
Vac+1 mth 12(10–14) 62 14(12–17) 56 0.11
Vac+6.5 mths 15(12–20) 76 15(11–21) 63 0.98
IL2 ex vivo ELISPOT: TH2R region
Screen 16(13–19) 107 16(13–20) 89 0.93
Vac+1 mth 13(10–17) 62 21(16–28) 56 0.003**
Vac+6.5 mths 18(14–23) 76 17(13–22) 63 0.76
IL2 ex vivo ELISPOT: TH3R/ CS.T3T region
Screen 17(15–21) 107 17(14–21) 89 0.9
Vac+1 mth 15(12–19) 62 24(19–31) 56 0.003**
Vac+6.5 mths 18(13–24) 76 29(20–41) 62 0.022*
IL2 ex vivo ELISPOT: All peptides summed
Screen 44(37–53) 107 42(34–51) 89 0.71
Vac+1 mth 35(27–45) 62 56(44–73) 56 0.002**
Vac+6.5 mths 45(34–60) 76 53(39–72) 62 0.43
*for p,0.05 or ** for p,0.005 where Mean for Rabies group , Mean for RTS,S/AS01E group. # for p,0.05 or ## for p,0.005 where Mean for Rabies group .
Mean for RTS,S/AS01E group.doi:10.1371/journal.pone.0025786.t002
Table 2. Cont.
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25786
Time course of responses (ICS assays)The frequencies of IL2, TNFa and IFNc producing CD4+ T
cells by ICS was significantly higher at one month after the final
vaccination with RTS,S/AS01E compared with pre-vaccination
levels (p,0.0001, p,0.0001, p = 0.0006, respectively). There was
then a fall in responses between 1 month and 12 months post
vaccination, falling to pre-vaccination levels for IL2 (p,0.0001)
and TNFa (p,0.0001). IFNc producing CD4+ T cells remained
above pre-vaccination levels, albeit at low frequency throughout.
However, there was an even more pronounced fall in CD4+ T cell
responses among control vaccinees (Table 2), and so RTS,S/
AS01E vaccinees had substantially higher T cell responses than
control vaccinees at 12 months post vaccination (p,0.0001 for
TNFa and IL2, p = 0.009 for IFNc).
Inter assay correlationsThere were strong correlations between the different cytokines
detected by ICS, and also between IL2/IFNc ELISPOT results
one month after vaccination with RTS,S/AS01E (Table 3).
Cultured ELISPOTs were significantly associated with ICS results,
but not with ex vivo ELISPOT results (IFNc or IL2). Antibody titres
were associated with all the cellular assays except ex vivo IFNcELISPOTs (Table 3).
Correlates of immunityAfter vaccination with RTS,S/AS01E, an increasing frequency
of TNFa producing, CS-specific CD4+ cells detected using ICS
was associated with a reduced risk of clinical malaria (HR = 0.64
for each 10 fold increase in the frequency of CD4+ TNFa+ T cells,
95%CI 0.49–0.86, p = 0.002). On ICS, IFNc production by CS-
specific CD4+ T cells was associated with a reduced risk of clinical
malaria of borderline significance (p = 0.07, Table 4). TNFa and
IFNc producing CS-specific CD4+ T cells were at much lower
frequencies among control vaccinees, but nevertheless were
associated with reduced risks of clinical malaria of borderline
significance. When data from both RTS,S/AS01E vaccinees and
control vaccinees were combined (with adjusting for vaccination
group), the overall hazard ratios were 0.74 (95%CI 0.62–0.89,
p = 0.001) and 0.79 (95%CI 0.67–0.94, p = 0.007) for TNFa and
IFNc, respectively. On Bonferroni adjustment, these p values were
0.018 and 0.13, respectively. Similar results were observed when
adjusting for anti-CSP antibody titres as a continuous variable
(HR = 0.75, 95%CI 0.62–0.91, p = 0.003 with p = 0.054 after
Bonferroni adjustment, and HR = 0.81, 95%CI 0.68–0.95,
p = 0.01 with p = 0.13 after Bonferroni adjustment for TNFaCD4+ T cells and IFNc CD4+ T cells, respectively).
In order to display the effect graphically, the cellular responses
were split by tertile (Figure 4). The middle tertiles for TNFa are at
intermediate risk, suggesting a continuous change in risk as the
frequency of TNFa cells increases rather than a threshold effect.
When the frequencies of TNFa producing CD4+ T cells and
IFNc producing CD4+ T cells were combined in the same model,
TNFa CD4+ T cell frequency was the independent factor (i.e.
HR = 0.76, 95%CI 0.64–0.89, p = 0.001 compared with
HR = 0.84, 95%CI 0.71–1.00, p = 0.050 for the frequency of
IFNc producing CD4+ T cells). An interaction term generated by
multiplying the frequency of TNFa CD4+ T cells by antibody
concentrations was not significant in determining risk (HR = 0.79,
95%CI 0.51–1.2, p = 0.29).
On applying the fourth of the Prentice criteria, we found that
vaccination group was still an independent predictor of clinical malaria
risk in a multivariable model including CD4+ TNFa+ cells
(HR = 0.69, 95%CI 0.48–0.97, p = 0.036). In other words, only 42%
of the effect of vaccination could be accounted for by CD4+ TNFa+cells. However, when anti-CS titers were added to the model the effect
of vaccine became non-significant (HR = 0.93, 95%CI 0.62–1.42,
p = 0.76, i.e. 87% of the effect of vaccination was accounted for).
Hence, while neither CD4+ TNFa+ cells nor anti-CS antibodies
alone accounted for all of the effect of vaccination with RTS,S/
AS01E on clinical malaria risk, the combination of CD4+ TNFa+T cells and anti-CS antibodies together could account for all of the
statistical effect of vaccination.
Discussion
Vaccination with RTS,S/AS01E induced circumsporozoite
protein (CS) specific T cell responses in 5–17 month-old children
living in a malaria endemic area. The frequency of CD4+ TNFa+ T
cells on ICS was associated with protection from clinical malaria.
Although the use of 15-mer peptides may have been sub-optimal to
demonstrate CD8+ T cell responses, we did in fact identify both
CD4+ and CD8+ responses above negative control conditions for
both RTS,S/AS01E and control vaccinees. However, the CD8+responses were apparently not induced by vaccination, and
presumably are the result of exposure to malaria parasites [32].
If TNFa producing CD4+ T cells are causally related to
protection, they should be associated with protection whether they
are acquired by vaccination or by natural exposure to malaria
Table 3. Inter-assay Correlation coefficients of CMI assays at 1 month post vaccination with RTS,S/AS01E.
CD4+ IFNc CD4+ IL2 CD4+ TNFa Antibody (CS) Cultured IFNc IFNc ELISPOT IL2 ELISPOT
CD4+ IFNc 1
CD4+ IL2 0.38*** 1
CD4+ TNFa 0.32*** 0.66*** 1
Antibody (CS) 0.14*** 0.35*** 0.26*** 1
Cultured IFNc 0.14** 0.15** 0.18*** 0.22*** 1
IFNc ELISPOT 20.05 20.05 0.02 0.03 20.02 1
IL2 ELISPOT 0 0.01 0.15* 0.11* 20.06 0.31*** 1
* = p,0.05.** = p,0.001.*** = p,0.0001.doi:10.1371/journal.pone.0025786.t003
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e25786
Figure 2. The time course of anti-CS CD4+ ICS responses and summed ELISPOT responses is shown per time point for RTS,S/AS01E
and control vaccination groups. * indicates p,0.05 and ** indicates p,0.005.doi:10.1371/journal.pone.0025786.g002
Figure 3. ELISPOT responses are shown for the individual stimulating peptide pools at 1 month post vaccination with RTS,S/AS01E.doi:10.1371/journal.pone.0025786.g003
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25786
parasites. Indeed, a borderline statistical correlation between
TNFa+ CD4+ T cells and a reduced risk of clinical malaria was
observed among control vaccinees. However, the overall level of
protection afforded by T cells among RTS,S/AS01E vaccinees was
greater, since the vaccinated children had 2–3 fold more TNFa+CD4+ T cells at 1 month and 8–10 fold more at 12 months post
vaccination. The frequency of TNFa_CD4+ T cells at 12 months
post vaccination was below screening (i.e. pre-vaccination) levels,
despite the fact that clinical protection was sustained at 15 months
[10]. Since clinical protection is determined by contemporaneous
comparison with control vaccinees, it is therefore theoretically
possible that the relative difference between RTS,S/AS01E
vaccinees and control vaccines is relevant despite this fall.
T cell responses to CSP rose between screening and 1 month
post-vaccination, and then fell to lower levels at 12 months post
vaccination. This was seen in both ELISPOT and ICS studies, and
could not be explained by greater background responses (which did
not change over time and were subtracted from antigen-specific
responses), or by non-specific responses detected in changing
positive controls over time. The temporary increase in CSP specific
responses parallels the increase and decrease in antibodies to blood
stage antigens seen in the same children [33]. Antibody responses
made by young children to blood stage antigens are often short-lived
[34], and may reflect short-term changes in exposure [35]. Taking
together the antibody data, the consistent pattern in ICS and
ELISPOT studies, the stability of positive and negative control
responses, and the accounting for background reactivity by
subtracting negative control responses from antigen-specific re-
sponses, show no significant variation over time for positive and
negative controls, we conclude that the T cell responses are raised
by exposure to malaria during the transmission season, but are
short-lived and therefore not sustained once exposure falls.
We did not identify an association between protection and T
cell responses detected by the cultured IFNc assay, as previously
reported [20]. However, we tested pools of peptides rather than
individual peptides, and the previously reported association was
specific to the CS.T3T peptide. The CS.T3T peptide was
contained in a pool of TH3R/CS.T3T peptides. In other studies,
the CS.T3T peptide accounted for more than half of the overall
response seen in the TH3R/CS.T3T peptide pool [36].
Furthermore, we identified very little IFNc production in our
study. Previous studies showing marked IFNc production have
been done in adults [18], and IFNc production may be suppressed
in children in malaria endemic areas [37].
Ex vivo ELISPOT studies did not correlate with ICS studies for
the same cytokines, even though both use overnight stimulations,
and ICS results were 10-fold higher than ELISPOT results. This
may be partially explained by measuring ELISPOT assays per
million PBMC, whereas ICS is measured per million CD4+ T
cells. However, ex vivo and cultured ELISPOTs identify different
Table 4. The hazard ratio from Cox regression models (with 95% CI) for the outcome clinical malaria by CMI assays.
Both datasets Rabies Vaccinees RTS,S/AS01E Vaccinees
Assay HR (95%CI) p HR (95%CI) p HR (95%CI) p
ICS: CD4 cells
IFNc 0.79(0.67–0.94) 0.007 0.81(0.66–1.01) 0.058 0.77(0.59–1.02) 0.07
IL2 0.9(0.76–1.07) 0.23 0.97(0.78–1.22) 0.81 0.77(0.55–1.08) 0.13
TNFa 0.74(0.62–0.89) 0.001 0.8(0.62–1.03) 0.084 0.64(0.49–0.86) 0.002
ICS: CD8 cells
IFNc 1.07(0.91–1.25) 0.43 1.13(0.93–1.37) 0.21 0.93(0.68–1.27) 0.65
IL2 0.85(0.69–1.05) 0.13 0.92(0.7–1.21) 0.56 0.79(0.56–1.12) 0.19
TNFa 0.87(0.72–1.04) 0.12 0.83(0.66–1.05) 0.11 0.93(0.68–1.28) 0.66
IFNc cultured ELISPOT
NANP 1.01(0.65–1.57) 0.95 0.9(0.54–1.52) 0.7 1.08(0.44–2.68) 0.87
TH2R 0.76(0.51–1.14) 0.18 0.67(0.3–1.52) 0.34 0.79(0.49–1.27) 0.33
TH3R 0.94(0.67–1.32) 0.72 0.99(0.61–1.61) 0.97 0.86(0.53–1.39) 0.54
Sum 0.95(0.67–1.34) 0.77 0.92(0.54–1.57) 0.77 0.92(0.58–1.47) 0.73
IFNc ex vivo ELISPOT
NANP 1.61(1.01–2.55) 0.044 1.54(0.87–2.72) 0.14 1.38(0.62–3.08) 0.44
TH2R 1(0.59–1.69) 1 0.9(0.44–1.85) 0.78 1.1(0.49–2.45) 0.81
TH3R 1.62(1.04–2.52) 0.032 1.57(0.82–2.99) 0.17 1.4(0.75–2.64) 0.29
Sum 1.35(0.86–2.12) 0.2 1.32(0.73–2.4) 0.35 1.23(0.61–2.47) 0.57
IL2 ex vivo ELISPOT
NANP 1.18(0.56–2.51) 0.67 1(0.29–3.39) 0.99 1.38(0.46–4.17) 0.57
TH2R 0.57(0.23–1.45) 0.24 0.49(0.13–1.79) 0.28 0.81(0.2–3.2) 0.76
TH3R 0.94(0.45–1.97) 0.87 0.73(0.21–2.49) 0.61 0.97(0.39–2.42) 0.94
Sum 0.83(0.38–1.83) 0.65 0.81(0.25–2.56) 0.72 0.78(0.23–2.62) 0.69
HR = Hazard Ratio for each log (i.e. ten-fold) increase in frequency of T cells. Confidence intervals are 5–95%. HRs are adjusted by anti-CS antibody titre (in 2 groups),age, area of residence, ITN use and distance from the dispensary. NANP = NANP and conserved region peptides pool, TH2R = TH2R region peptides pool, TH3R = TH3Rand CS.T3T region peptide pool, Sum = all three peptide pools summed.doi:10.1371/journal.pone.0025786.t004
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25786
cell populations, the latter more closely reflecting a central
memory phenotype [38,39,40]. Hence it is possible that ICS and
cultured ELISPOT identify central memory cells, but ex vivo
ELISPOTs identify effector phenotypes. ELISPOT assays failing
internal control standards were excluded, but similar control
standards were not pre-defined for ICS data. However, this
applied to a minority of assays (2% and 5%, respectively for ex vivo
IFNc and IL2 ELISPOTS) which did not correlate with ICS data,
and 8% for cultured ELISPOTS, which did correlate. Hence it
seems unlikely that lack of quality control standards for ICS
explained the lack of correlation.
The CD4+ T cell response associated with protection in our
analysis (i.e. TNFa production) were at a low frequency (mean 426
cells per million CD4 cells at peak). Higher frequency responses
have been required to achieve protection in sporozoite challenge
studies [7]. However, the antibody concentrations associated with
protection are also higher in sporozoite challenge studies [7,10].
These differences in outcome may be explained by the greater
sporozoite inoculum used in challenge studies compared with
exposure in the field [41]. The IFNc response that was apparently
associated with protection in our study was very low frequency and
barely above the limit of detection (25 cells per million), and the
apparent association is likely to reflect the association between
IFNc and TNFa rather than an independent effect. We were
unable to assess polyfunctionality in the present study, because the
high number of samples (n = 1066, with three conditions per
sample) required an automated gating strategy using FACSDiva
software (BD Biosciences). The FACSdiva analysis used for this
study did not include Boolean gates in order to obtain
polyfunctional T cell data. Further analysis to determine
polyfunctionality in this large dataset is ongoing.
Multiple comparisons (i.e. 18) have been undertaken to identify
the association between TNFa producing CD4+ T cells and
protection from clinical malaria. However, the association was
highly significant (p = 0.001) and remains significant after a
Bonferroni correction (p = 0.018).
The Prentice criteria have been proposed as a way of qualifying
surrogate endpoints [42,43] and include four criteria [30]. We
found that the combination of anti-CS titers and TNFa producing
CD4+ T cells met all the criteria (i.e. vaccination was associated
with protection; anti-CS titers and CD4+ TNFa+ T cells were
both independently associated with vaccination; were both
Figure 4. Survival plots with time to first episode of clinical malaria plotted for RTS,S/AS01E (left columns) and control vaccinees(left and right columns) according to tertile of CD4+, TNFa responses (top row), CD4+ IFNc responses (middle row) and IFNc ex vivoELISPOT responses to TH3R/CS.T3T peptides pool (lower row). Where more than one third of responses were at the lower limit of detection,the lower two tertiles are combined (and hence only 2 tertiles are displayed on some plots). For CD4+ TNFa+ responses, the tertiles were 1 to 154(lower), 155 to 407 (middle) and 408 to 28,840 (upper) cells per million for RTS,S/AS01E vaccinees, and 1 to 26 (lower), 27 to 165 (middle) and 166 to10,000 (upper) cells per million for control vaccinees. For CD+ IFNc+ responses the tertiles were 1 to 12 (lower), 13 to 66 (middle) and 67 to 8,320(upper) cells per million for RTS,S/AS01E vaccinees, and 1 to 40 (lower) and 41 to 5,980 (upper) cells per million for rabies vaccinees. The time point ‘‘0months’’ refers to the time of a blood draw. Cellular responses were analyzed as time-varying covariates, where the effect of cellular responses fromall available blood draws was related to clinical malaria episodes during the period of monitoring after each measurement. Therefore, each RTS,Svaccinee could contribute to 2 periods of monitoring. These three assays were selected for the figure because significant associations on Coxregression were seen (Table 4).doi:10.1371/journal.pone.0025786.g004
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e25786
independently associated with protection; and the combination of
anti-CS titers and CD4+ TNFa+ T cells, but not either alone,
could account for the effect of vaccination in multi-variable Cox
regression analysis). We found no significant interaction between
anti-CS titers and TNFa+ T cells.
Microheterogeneity of malaria exposure has been observed in
Kilifi [44], and may confound the association between antibodies
to blood stage malaria antigens and the risk of malaria [33].
However, this is unlikely to explain the association between CD4+TNFa+ T cells and protection from clinical malaria for two
reasons: The direction of confounding was in the opposite
direction in this cohort (i.e. microheterogeneity led to a
confounded association between increasing antibody levels and
increasing risk of malaria rather than protection), and the
association with protection is more marked in RTS,S/AS01E
vaccinees rather than control vaccinees.
There is strong evidence that anti-CS antibodies inhibit
sporozoite invasion [45], supporting a causal relationship, and
TNFa may reduce the parasite’s intrahepatocytic development
[13,14]. However, it is possible that the frequency of CD4+TNFa+ T cells is associated with another causal mediator of
immunity (for instance better quality antibody responses, en-
hanced T cell memory or polyfunctionality). These further
characterizations of the immune response should now be a
priority, since establishing an immunological surrogate endpoint
will accelerate the development of candidate malaria vaccines,
inform monitoring the persistence of immune responses and
inform the timing of a booster dose.
Supporting Information
Supporting Information S1
(PPT)
Acknowledgments
We are grateful to the parents of participants and village and district
authorities for their cooperation; the Data and safety monitoring board,
chaired by Prof. Malcolm Molyneux, and the local safety monitor, Dr. Jay
Berkley in Kilifi; Edna Ogada, Juliana Wambua (site coordinator), and
Dorothy Mwachiro (community liaison officer) for providing support in
Kilifi; and the staff of the Malaria Project Team at GlaxoSmithKline — in
particular, Mickael Mestre (technical assistance), Olivier Jauniaux
(technical assistance), Robbert Van-der-Most and Lode Schuerman for
pre-submission reviews of the manuscript, Nathalie Annez, Sarah Benns
(professional writer), Srilakshmi Pranesh, Marie-Sylvie Remacle (profes-
sional writer) and Laurence Vigneron.
Author Contributions
Conceived and designed the experiments: PM JV ML AL TV BS KM JC
PB. Performed the experiments: AO PM JM DK MJ OK EJ PB. Analyzed
the data: AO ML PB. Wrote the paper: AO JV AL JC PB.
References
1. Ballou WR (2009) The development of the RTS,S malaria vaccine candidate:
challenges and lessons. Parasite Immunol 31: 492–500.
2. Lell B, Agnandji S, von Glasenapp I, Haertle S, Oyakhiromen S, et al. (2009) A
randomized trial assessing the safety and immunogenicity of AS01 and AS02 adjuvanted
RTS,S malaria vaccine candidates in children in Gabon. PLoS One 4: e7611.
3. Bojang KA, Olodude F, Pinder M, Ofori-Anyinam O, Vigneron L, et al. (2005)
Safety and immunogenicty of RTS,S/AS02A candidate malaria vaccine in
Gambian children. Vaccine 23: 4148–4157.
4. Bojang KA, Milligan PJ, Pinder M, Vigneron L, Alloueche A, et al. (2001)
Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum
infection in semi-immune adult men in The Gambia: a randomised trial. Lancet
358: 1927–1934.
5. Doherty JF, Pinder M, Tornieporth N, Carton C, Vigneron L, et al. (1999) A
phase I safety and immunogenicity trial with the candidate malaria vaccine
RTS,S/SBAS2 in semi-immune adults in The Gambia. Am J Trop Med Hyg
61: 865–868.
6. Stoute JA, Slaoui M, Heppner DG, Momin P, Kester KE, et al. (1997) A
preliminary evaluation of a recombinant circumsporozoite protein vaccine
against Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation
Group. N Engl J Med 336: 86–91.
7. Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, et al.
(2009) Randomized, double-blind, phase 2a trial of falciparum malaria vaccines
RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and
immunologic associates of protection. J Infect Dis 200: 337–346.
8. Guinovart C, Aponte JJ, Sacarlal J, Aide P, Leach A, et al. (2009) Insights into
long-lasting protection induced by RTS,S/AS02A malaria vaccine: further
results from a phase IIb trial in Mozambican children. PLoS One 4: e5165.
9. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, et al. (2004) Efficacy of the
RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in
young African children: randomised controlled trial. Lancet 364: 1411–1420.
10. Olotu A, Lusingu J, Leach A, Lievens M, Vekemans J, et al. (2011) Efficacy of
RTS,S/AS01E malaria vaccine and exploratory analysis on anti-circumspor-
ozoite antibody titres and protection in children aged 5-17 months in Kenya and
Tanzania: a randomised controlled trial. Lancet Infect Dis 11: 102–109.
11. Wang R, Charoenvit Y, Corradin G, De La Vega P, Franke ED, et al. (1996)
Protection against malaria by Plasmodium yoelii sporozoite surface protein 2
linear peptide induction of CD4+ T cell- and IFN-gamma-dependent
elimination of infected hepatocytes. J Immunol 157: 4061–4067.
12. McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, et al. (2003)
Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by
recombinant modified vaccinia virus Ankara in humans. Nat Med 9: 729–735.
13. Butler NS, Schmidt NW, Harty JT (2010) Differential effector pathways regulate
memory CD8 T cell immunity against Plasmodium berghei versus P. yoelii
sporozoites. J Immunol 184: 2528–2538.
14. Depinay N, Franetich JF, Gruner AC, Mauduit M, Chavatte JM, et al. (2011)
Inhibitory effect of TNF-alpha on malaria pre-erythrocytic stage development:
influence of host hepatocyte/parasite combinations. PLoS ONE 6: e17464.
15. Doolan DL, Hoffman SL (2000) The complexity of protective immunity against
liver-stage malaria. J Immunol 165: 1453–1462.
16. Casares S, Brumeanu TD, Richie TL (2010) The RTS,S malaria vaccine.
Vaccine 28: 4880–4894.
17. Moorthy VS, Ballou WR (2009) Immunological mechanisms underlying
protection mediated by RTS,S: a review of the available data. Malar J 8: 312.
18. Lalvani A, Moris P, Voss G, Pathan AA, Kester KE, et al. (1999) Potent
induction of focused Th1-type cellular and humoral immune responses by
RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine. J Infect
Dis 180: 1656–1664.
19. Stoute JA, Kester KE, Krzych U, Wellde BT, Hall T, et al. (1998) Long-term
efficacy and immune responses following immunization with the RTS,S malaria
vaccine. J Infect Dis 178: 1139–1144.
20. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, et al. (2004) A
CD4(+) T-cell immune response to a conserved epitope in the circumsporozoite
protein correlates with protection from natural Plasmodium falciparum infection
and disease. Nat Med 10: 406–410.
21. Pinder M, Reece WH, Plebanski M, Akinwunmi P, Flanagan KL, et al. (2004)
Cellular immunity induced by the recombinant Plasmodium falciparum malaria
vaccine, RTS,S/AS02, in semi-immune adults in The Gambia. Clin Exp
Immunol 135: 286–293.
22. Sun P, Schwenk R, White K, Stoute JA, Cohen J, et al. (2003) Protective
immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium
falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells
producing IFN-gamma. J Immunol 171: 6961–6967.
23. Barbosa A, Naniche D, Aponte JJ, Manaca MN, Mandomando I, et al. (2009)
Plasmodium falciparum-specific cellular immune responses after immunization
with the RTS,S/AS02D candidate malaria vaccine in infants living in an area of
high endemicity in Mozambique. Infect Immun 77: 4502–4509.
24. Olotu AI, Fegan G, Bejon P (2010) Further analysis of correlates of protection
from a phase 2a trial of the falciparum malaria vaccines RTS,S/AS01B and
RTS,S/AS02A in malaria-naive adults. J Infect Dis 201: 970–971.
25. Bejon P, Lusingu J, Olotu A, Leach A, Lievens M, et al. (2008) Efficacy of
RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N
Engl J Med 359: 2521–2532.
26. Ansong D, Asante KP, Vekemans J, Owusu SK, Owusu R, et al. (2011) T cell
responses to the RTS,S/AS01(E) and RTS,S/AS02(D) malaria candidate
vaccines administered according to different schedules to Ghanaian children.
PLoS ONE 6: e18891.
27. Agnandji ST, Fendel R, Mestre M, Janssens M, Vekemans J, et al. (2011)
Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e25786
in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D).
PLoS ONE 6: e18559.
28. Plotkin SA (2008) Vaccines: correlates of vaccine-induced immunity. Clin Infect
Dis 47: 401–409.
29. Qin L, Gilbert PB, Corey L, McElrath MJ, Self SG (2007) A framework for
assessing immunological correlates of protection in vaccine trials. J Infect Dis
196: 1304–1312.
30. Prentice RL (1989) Surrogate endpoints in clinical trials: definition and
operational criteria. Stat Med 8: 431–440.
31. Macete EV, Sacarlal J, Aponte JJ, Leach A, Navia MM, et al. (2007) Evaluation
of two formulations of adjuvanted RTS, S malaria vaccine in children aged 3 to
5 years living in a malaria-endemic region of Mozambique: a Phase I/IIb
randomized double-blind bridging trial. Trials 8: 11.
32. Flanagan KL, Lee EA, Gravenor MB, Reece WH, Urban BC, et al. (2001)
Unique T cell effector functions elicited by Plasmodium falciparum epitopes in
malaria-exposed Africans tested by three T cell assays. J Immunol 167:
4729–4737.
33. Bejon P, Cook J, Bergmann-Leitner E, Olotu A, Lusingu J, et al. (2011) Effect of
the Pre-erythrocytic Candidate Malaria Vaccine RTS,S/AS01E on Blood Stage
Immunity in Young Children. J Infect Dis 204: 9–18.
34. Kinyanjui SM, Bull P, Newbold CI, Marsh K (2003) Kinetics of antibody
responses to Plasmodium falciparum-infected erythrocyte variant surface
antigens. J Infect Dis 187: 667–674.
35. Bejon P, Turner L, Lavstsen T, Cham G, Olotu A, et al. (2011) Serological
Evidence of Discrete Spatial Clusters of Plasmodium falciparum Parasites. PLoS
ONE 6: e21711.
36. Dunachie SJ, Walther M, Vuola JM, Webster DP, Keating SM, et al. (2006) A
clinical trial of prime-boost immunisation with the candidate malaria vaccines
RTS,S/AS02A and MVA-CS. Vaccine 24: 2850–2859.
37. Bejon P, Mwacharo J, Kai O, Todryk S, Keating S, et al. (2007) The induction
and persistence of T cell IFN-gamma responses after vaccination or naturalexposure is suppressed by Plasmodium falciparum. J Immunol 179: 4193–4201.
38. Godkin AJ, Thomas HC, Openshaw PJ (2002) Evolution of epitope-specific
memory CD4(+) T cells after clearance of hepatitis C virus. J Immunol 169:2210–2214.
39. Keating SM, Bejon P, Berthoud T, Vuola JM, Todryk S, et al. (2005) DurableHuman Memory T Cells Quantifiable by Cultured Enzyme-Linked Immuno-
spot Assays Are Induced by Heterologous Prime Boost Immunization and
Correlate with Protection against Malaria. J Immunol 175: 5675–5680.40. Todryk SM, Pathan AA, Keating S, Porter DW, Berthoud T, et al. (2009) The
relationship between human effector and memory T cells measured by ex vivo andcultured ELISPOT following recent and distal priming. Immunology 128: 83–91.
41. Bejon P, Andrews L, Andersen RF, Dunachie S, Webster D, et al. (2005)Calculation of liver-to-blood inocula, parasite growth rates, and preerythrocytic
vaccine efficacy, from serial quantitative polymerase chain reaction studies of
volunteers challenged with malaria sporozoites. J Infect Dis 191: 619–626.42. Kohberger RC, Jemiolo D, Noriega F (2008) Prediction of pertussis vaccine
efficacy using a correlates of protection model. Vaccine 26: 3516–3521.43. Ray ME, Bae K, Hussain MH, Hanks GE, Shipley WU, et al. (2009) Potential
surrogate endpoints for prostate cancer survival: analysis of a phase III
randomized trial. J Natl Cancer Inst 101: 228–236.44. Bejon P, Williams TN, Liljander A, Noor AM, Wambua J, et al. (2010) Stable
and unstable malaria hotspots in longitudinal cohort studies in Kenya. Plos Med7: e1000304.
45. Hollingdale MR, Appiah A, Leland P, do Rosario VE, Mazier D, et al. (1990)Activity of human volunteer sera to candidate Plasmodium falciparum
circumsporozoite protein vaccines in the inhibition of sporozoite invasion assay
of human hepatoma cells and hepatocytes. Trans R Soc Trop Med Hyg 84:325–329.
T Cell Immunity and RTS,S/AS01E
PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e25786
